Myofibroblastic transdifferentiation of quiescent Ito cells is characteristic of alcohol-induced fibrosis and is responsible for increased production of extracellular matrix; however, factors initiating Ito cell activation are unknown. The overall goal of this proposal is to delineate the mechanism(s) by which CYP2E1- expressing hepatocytes initiate transdifferentiation of quiescent Ito cells to myofibroblasts. The first Specific Aim is designed to determine the role of CYP2E1-mediated lipid peroxidation in triggering cytokine release from stressed hepatocytes. This will be accomplished by producing a non-transformed hepatocyte cell line that stably over-expresses CYP2E1. Oxidative stress will be assessed by measuring alcohol induced glutathione depletion and unsaturated aldehyde liberation. Cytokines produced in response to oxidative stress will be identified. In situ hybridization will demonstrate expression of identified cytokine genes in ethanol-fed rats. The second aim is to characterize the activation of quiescent Ito cells by CYP2E1-expressing hepatocytes, and to investigate the contribution of Kupffer cells to hepatocyte-mediated Ito cell activation. Transdifferentiation will be investigated utilizing a culture system that does not produce Ito cell culture-activation. The third aim will describe the cytokine dependent signal transduction pathways which stimulate quiescent Ito cells transformation to the myofibroblast phenotype. The precise temporal relationship between initiation of Ito cell transdifferentiation, transcription factor activation and cytokine signal transduction pathway activation will be explored. The in vivo relationship between cytokine production, Ito cell activation and cytokine receptor expression will be described.